Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme |
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| Authors: | Giuseppe Grasso Ashley I Bush Roberta D’Agata Enrico Rizzarelli Giuseppe Spoto |
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| Institution: | (1) Dipartimento Scienze Chimiche, Università di Catania, v.le A. Doria 6, 95125 Catania, Italy;(2) Department of Psychiatry, Massachusetts General Hospital, Charlestone, MA, USA;(3) Department of Pathology, Mental Health Research Institute of Victoria, The University of Melbourne, Parkville, Australia;(4) Istituto di Biostrutture e Bioimmagini, CNR, v.le A. Doria 6, 95125 Catania, Italy |
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| Abstract: | Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored
on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays.
In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze
the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS
combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability
to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity
of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis.
The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and
insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained. |
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| Keywords: | Solid-state assay Surface plasmon resonance Mass spectrometry Insulin degrading enzyme Conformational change |
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